A stable flank of unstable lymphotropic papovavirus integration sites is associated with a cellular S1 nuclease-sensitive sequence

Abstract Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

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S1 nuclease mapping analysis of ribosomal RNA processing in wild type and processing deficient Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44336-5 ◽

1983 ◽

Vol 258 (19) ◽

pp. 12034-12042

Author(s):

T C King ◽

D Schlessinger

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Rna Processing ◽

Wild Type ◽

S1 Nuclease ◽

Mapping Analysis ◽

S1 Nuclease Mapping ◽

Ribosomal Rna Processing ◽

Nuclease Mapping

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Stabilization of telomeric G-quadruplex by ligand binding increases susceptibility to S1 nuclease

Chemical Communications ◽

10.1039/d1cc03294a ◽

2021 ◽

Author(s):

Ryo Ishikawa ◽

Mizuho Yasuda ◽

Shogo Sasaki ◽

Yue Ma ◽

Kazuo Nagasawa ◽

...

Keyword(s):

Ligand Binding ◽

S1 Nuclease ◽

G Quadruplex ◽

Thermodynamic Stabilization

The extent of thermodynamic stabilization of telomeric G-quadruplex (G4) by isomers of G4 ligand L2H2-6OTD, a telomestatin analog, is inversely correlated with susceptibility to S1 nuclease. L2H2-6OTD facilitated the S1...

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213. Detection of Integration Sites of CD19 Chimeric Antigen Receptor Gene in a Non-Viral Gene Transfer System

Molecular Therapy ◽

10.1016/s1525-0016(16)33022-2 ◽

2016 ◽

Vol 24 ◽

pp. S83-S84

Author(s):

Motoharu Hamada

Keyword(s):

Gene Transfer ◽

Chimeric Antigen Receptor ◽

Receptor Gene ◽

Antigen Receptor ◽

Viral Gene ◽

Transfer System ◽

Viral Gene Transfer ◽

Integration Sites ◽

Gene Transfer System

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Circular Double-Stranded Forms of TT Virus DNA in the Liver

Journal of Virology ◽

10.1128/jvi.74.11.5161-5167.2000 ◽

2000 ◽

Vol 74 (11) ◽

pp. 5161-5167 ◽

Cited By ~ 70

Author(s):

Hiroaki Okamoto ◽

Masato Ukita ◽

Tsutomu Nishizawa ◽

Junichi Kishimoto ◽

Yuji Hoshi ◽

...

Keyword(s):

Human Beings ◽

Specific Primer ◽

Dna Virus ◽

S1 Nuclease ◽

Tt Virus ◽

Double Stranded Dna ◽

Replicative Intermediates ◽

Paired Serum ◽

Genomic Length ◽

Liver Tissues

ABSTRACT TT virus (TTV) is an unenveloped, circular, and single-stranded DNA virus commonly infecting human beings worldwide. TTV DNAs in paired serum and liver tissues from three viremic individuals were separated by gel electrophoresis and characterized biophysically. TTV DNAs in sera migrated in sizes ranging from 2.0 to 2.5 kb. TTV DNAs in liver tissues, however, migrated at 2.0 to 2.5 kb as well as at 3.5 to 6.1 kb. Both faster- and slower-migrating forms of TTV DNAs in the liver were found to be circular and of the full genomic length of 3.8 kb. TTV DNAs migrating at 2.0 to 2.5 kb, from either serum or liver tissues, were sensitive to S1 nuclease but resistant to restriction endonucleases, and therefore, they were single-stranded. By contrast, TTV DNAs in liver tissues that migrated at 3.5 to 6.1 kb were resistant to S1 nuclease. They migrated at 3.7 to 4.0 kb after digestion with EcoRI, which suggests that they represent circular, double-stranded replicative intermediates of TTV. When TTV DNAs were subjected to strand-specific primer extension and then amplified by PCR with internal primers, those in serum were found to be minus-stranded DNAs while those in liver tissues were found to be a mixture of plus- and minus-stranded DNAs. These results suggest that TTV replicates in the liver via a circular double-stranded DNA.

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Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽

10.1139/g91-098 ◽

1991 ◽

Vol 34 (4) ◽

pp. 644-651 ◽

Cited By ~ 16

Author(s):

Kenneth Koo ◽

W. Dorsey Stuart

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Neurospora Crassa ◽

Rna Binding ◽

Signal Sequence ◽

Average Length ◽

Open Reading Frame ◽

Mrna Transcript ◽

S1 Nuclease ◽

Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

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